Characterization of rabbit antisera elicited with human LMP2- and LMP7-specific peptides and recombinant proteins.
Anti-human LMP2 and anti-human LMP7 sera with a titer of at least 1:10,000 were developed by immunizing rabbits with LMP2- and LMP7-specific peptides corresponding to C-terminal regions of each subunit or with TrxLMP2 and TrxLMP7 recombinant proteins. IgG antibodies elicited by immunization with LMP-specific peptides or recombinant proteins displayed reactivity with their respective immunogens in ELISA. Furthermore, antibodies elicited with both types of immunogens recognize native and recombinant LMP2 and LMP7 subunits in Western blotting and are able to immunoprecipitate LMP2 and LMP7 as components of the 20S proteasome from lymphoid cell lysates. In ELISA, a subpopulation of the antibodies generated with LMP peptides and recombinant proteins corresponding to one LMP subunit is cross-reactive with the other one. This antibody subpopulation was not detectable in the affinity-purified antibody populations isolated by passing antisera over the corresponding immunogen. Neither anti-LMP2 nor anti-LMP7 sera displayed cross-reactivity with the homologous proteasome subunits Delta and MB1. In immunohistochemical reactions affinity-purified anti-LMP2 and anti-LMP7 antibodies stained cells in both frozen and formalin-fixed tissue sections of normal skin. These results indicate that the anti-LMP2 and anti-LMP7 sera elicited with peptides and recombinant proteins are both useful reagents for biochemical characterization of LMP2 and LMP7 and to analyze their expression in normal and transformed cells.
Dellaratta
D V
DV
Department of Microbiology and Immunology, New York Medical College, Valhalla 10595, USA.
Hicklin
D J
DJ
Kishore
R
R
Kageshita
T
T
Ferrone
S
S
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D002460:Cell Line; D003546:Cysteine Endopeptidases; D005260:Female; D006801:Humans; D008285:Major Histocompatibility Complex; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology*; D046988:Proteasome Endopeptidase Complex; D011506:Proteins / Q000235:genetics / Q000276:immunology*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012867:Skin / Q000276:immunology / Q000473:pathology; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; C071313:LMP-2 protein; D003546:Cysteine Endopeptidases; C077436:LMP7 protein; D046988:Proteasome Endopeptidase Complex